1. Field of the Invention
This invention relates to sequential chemical reactions of which the polymerase chain reaction (PCR) is one example. In particular, this invention addresses the methods and apparatus for performing chemical reactions simultaneously in a multitude of reaction media and independently controlling the reaction in each medium.
2. Description of the Prior Art
PCR is one of many examples of chemical processes that require precise temperature control of reaction mixtures with rapid temperature changes between different stages of the procedure. PCR itself is a process for amplifying DNA, i.e., producing multiple copies of a DNA sequence from a single strand bearing the sequence. PCR is typically performed in instruments that provide reagent transfer, temperature control, and optical detection in a multitude of reaction vessels such as wells, tubes, or capillaries. The process includes a sequence of stages that are temperature-sensitive, different stages being performed at different temperatures and the temperature being cycled through repeated temperature changes.
While PCR can be performed in any reaction vessel, multi-well reaction plates are the reaction vessels of choice. In many applications, PCR is performed in “real-time” and the reaction mixtures are repeatedly analyzed throughout the process, using the detection of light from fluorescently-tagged species in the reaction medium as a means of analysis. In other applications, DNA is withdrawn from the medium for separate amplification and analysis. In multiple-sample PCR processes in which the process is performed concurrently in a number of samples, a preferred arrangement is one in which each sample occupies one well of a multi-well plate or plate-like structure, and all samples are simultaneously equilibrated to a common thermal environment at each stage of the process. In some cases, samples are exposed to two thermal environments to produce a temperature gradient across each sample.
In the typical PCR instrument, a 96-well plate with a sample in each well is placed in contact with a metal block that is heated and cooled either by a Peltier heating/cooling apparatus or by a closed-loop liquid heating/cooling system that circulates a heat transfer fluid through channels machined into the block. Certain instruments, such as the SMART CYCLER® II System sold by Cepheid (Sunnyvale, Calif., USA), provide different thermal environments in different reaction vessels by using individual reaction vessels or capillaries. These instruments are costly and unable to reliably achieve temperature uniformity. The Institute of Microelectronics, of Singapore, likewise offers an instrument that provides multiple thermal environments, but does so by use of an integrated circuit to create individual thermal domains. This method is miniaturized but does not allow the use of multi-well reaction plates, which are generally termed microplates.